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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
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REVIEW
Origin, Microbiology, Nutrition, and Pharmacology of d-Amino Acids 1)
by Mendel Friedman
Western Regional Research Center, Agricultural Research Service, USDA, Albany CA 94710, USA
(phone: þ 1-510-5595615; fax: þ 1-510-5595777; e-mail: [email protected])
Exposure of food proteins to certain processing conditions induces two major chemical changes:
racemization of all l-amino acids (LAAs) to d-amino acids (DAAs) and concurrent formation of crosslinked amino acids such as lysinoalanine (LAL). The diet contains both processing-induced and
naturally-formed DAA. The latter include those found in microorganisms, plants, and marine
invertebrates. Racemization impairs digestibility and nutritional quality. Racemization of LAA residues
to their d-isomers in food and other proteins is pH-, time-, and temperature-dependent. Although
racemization rates of LAA residues in a protein vary, relative rates in different proteins are similar. The
nutritional utilization of different DAAs varies widely in animals and humans. Some DAAs may exert
both adverse and beneficial biological effects. Thus, although d-Phe is utilized as a nutritional source of
l-Phe, high concentrations of d-Tyr in such diets inhibit the growth of mice. Both d-Ser and LAL induce
histological changes in the rat kidney. The wide variation in the utilization of DAAs is illustrated by the
fact that, whereas d-Meth is largely utilized as a nutritional source of the l-isomer, d-Lys is not. Similarly,
although l-CysSH has a sparing effect on l-Meth when fed to mice, d-CysSH does not. Since DAAs are
consumed as part of their normal diet, a need exists to develop a better understanding of their roles in
foods, microbiology, nutrition, and medicine. To contribute to this effort, this overview surveys our
present knowledge of the chemistry, nutrition, safety, microbiology, and pharmacology of DAAs. Also
covered are the origin and distribution of DAAs in food and possible roles of DAAs in human physiology,
aging, and the etiology and therapy of human diseases.
1. Introduction. – Microorganism-synthesized d-amino acids (DAAs) are estimated
to constitute approximately one third of the human DAA burden [1]. During food
processing, naturally occurring l-amino acids (LAAs) may be transformed to their
mirror-image configuration d-isomers. A better understanding of the dietary significance of DAAs requires knowledge about the factors that induce racemization of LAA
to DAA in food proteins during food processing. Processing-induced amino acid
racemization includes those formed during exposure of food proteins to high pH, heat,
and acids. The nutritional effectiveness of protein-bound essential DAAs depends on
the amino acid composition, digestibility, and physiological utilization of released
amino acids. Since an amino acid must be liberated by digestion before nutritional
utilization can occur, the decreased susceptibility to digestion by proteolytic enzymes of
d-d, d-l, and l-d peptide bonds in DAA-containing proteins is a major factor adversely
affecting the bioavailability of protein-bound DAAs. In addition, some DAAs may be
toxic. Some DAAs have been shown to impart beneficial effects in vivo. It is not known
1)
Proceedings of First International Conference of d-Amino Acid Research, Awaji Island, Japan, July
1 – 4, 2009.
2010 Verlag Helvetica Chimica Acta AG, Zrich
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whether the biological effects of DAAs vary, depending on whether they are consumed
in the free state or as part of protein that contains processing-induced DAAs. To crossfertilize information among several disciplines and to stimulate further needed studies,
this review attempts to integrate and correlate the widely scattered literature on the
origin, formation, chemistry, microbiology, nutrition, pharmacology, and toxicology,
and medical aspects of DAA. This review is intended to stimulate interest in further
research to optimize beneficial effects of DAAs.
Origin of Chirality. Although the formation of l-amino acid (LAA) and d-sugars in
the primordial environment during evolution of life is generally considered to be a
result of chance [2], definitive proof of the origin of homochirality is still elusive, as
illustrated by the following selected observations listed chronologically.
A mathematical model was used to describe the parity-violating energy
differences between enantiomeric molecules [3]. The model predicts that an
enantiomeric excess (ee) is expected in chiral products resulting from reactions of
achiral or racemic substrates. A non-equilibrium racemic reaction system such as the
polymerization of d,l-amino acids to a protein spontaneously switches to a homochiral
reaction channel, giving only the l-product. Calculations show that polypeptides of the
l-series are preferentially stabilized by electroweak interactions. Such polypeptides
have lower energies due to parity violation than the corresponding d-isomers. Since the
formation of optically active biomolecules may be energy-driven, the creation of
homochiral biochemistry may not be accidental.
Based on crystallization studies, Cintas [4] suggests that homochirality may be the
result of differences in energy and growth rates of crystal surfaces to which d- or lamino acids bind. The autocatalytic cycles during crystallization and oligomerization
may account for homochirality of living organisms.
A review of the literature on cycles of solvation, crystallization, and adsorption
processes in the primordial environments (geochromatography) suggests that very
small differences in the physical properties of some enantiomers may account for the
enhanced formation of pure l-enantiomers, as well as for the preferential incorporation
of LAAs and d-carbohydrates into early life forms [5].
Another plausible idea is that the asymmetric earth rotation caused (induced) at
dawn and dusk circularly polarized UV light of opposite polarity and reversed
temperature profiles in the oceans [6]. Destruction of the d-isomer by the polarized
UV light in a sea surface hotter than at dawn created a daily l-isomer excess protected
from radiation by nightfall. The LAAs are then preserved by mechanical diffusion into
cold, darker regions of the sea leading to an LAA excess in early marine life. The
authors suggest that these postulated events can be subjected to computer simulation.
Although the selection of d-ribose in RNA and d-deoxyribose leads to the
biosynthesis of proteins exclusively from l-amino acids [7], Bolik et al. [8] provide
experimental evidence for the preferential stabilization of the natural d- over the
nonnatural l-configuration in nucleic acids. The energy differences in electronic levels
of d- and l-RNA may have led to the preferential stabilization of the naturally
occurring d-RNA over the l-configuration.
Nonenzymatic aminoacylation of an RNA minihelix occurred preferentially with
l-amino acids [9]. This finding suggests that tRNA aminoacylation, a process by which
amino acids first encounter RNA, may have been a critical step in determining LAA
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homochirality. However, it should be noted that cells contain defenses against daminoacyl-tRNAs [10].
It is likely that more than one physical-chemical event in the primordial
environment occurred simultaneously or consecutively that resulted in the creation
of homochirality favoring LAAs. The creation of l-homochirality is not the end of
creation, however, because special needs seem to have induced nature to also devise
enzyme-catalyzed alternate biosynthetic pathways to DAAs [11] [12]. Enzymes that are
known to be involved in DAA synthesis and metabolism include DAA oxidases [13],
deacylases [14], dehydrogenases [15], epimerases, [16] [17], proteases [18], and
racemases [15].
Because DAAs from natural sources are part of the human diet, we will first briefly
review their formation and function in plants, microbes, and other natural sources,
followed by a discussion of factors that favor racemization by conditions encountered
during home and commercial processing of food.
2. Analysis. – Because all of the LAA residues (Fig. 1) in a protein and additional
formed during food processing (Fig. 2) can undergo racemization simultaneously, but
at differing rates, assessment of the extent of racemization in a protein is a difficult task
that requires quantitative measurement of ca. 40 l- and d-optical isomers [19].
Reported methods include the use of chiral-phase gas chromatography [20], HPLC
[21], HPLC with enzyme reactors [22], LC/MS [23] [24], capillary electrophoresis
[25] [26], electrokinetic chromatography with detection by laser-induced fluorescence
[27], and biosensors [28] [29]. Detailed discussion of these and other methods for
analysis of DAA in food and biological samples is beyond the scope of this review [30 –
40].
3. Natural Occurrence of DAAs. – Plants. Plants appear to be able to synthesize
DAA derivatives and peptides de novo [41 – 43], as illustrated by the following
examples. Germinating pea seedlings (Pisum sativum) contain high concentrations of
N-malonyl-d-Ala and g-l-glutamyl-d-Ala [44]. The concentration of the latter
increased to 2.5 mm/seedling after 8 d of germination. d-Ala-d-Ala was found to be
present in tobacco leaves [45]. The relative concentrations of three d-Ala peptides (dAla-Gly, d-Ala-d-Ala, and d-Ala-l-Ala) produced in rice plants (genus Oryzae)
appears to be related to the nature of the rice strain [46]. Approximately 7% of the
total alanine content in grass leaves consisted of d-Ala [47]. Exogenously supplied dAla, possibly of microbial origin, is required for the plant to synthesize the dipeptides.
Ono et al. [48] demonstrated the presence of alanine racemase and of DAAs in alfalfa
seedlings (Medicago sativa L.). Studies by Forsum et al. [49] indicate that transgenic
plants can be engineered to utilize DAAs from the soil as a nitrogen source. The plant
racemase resembled racemases of bacterial origin. Hydrolyzed proteins used as plant
fertilizers also contain significant amounts of DAAs [25].
3.1. Microbes. Microbes provide a potential source of dietary DAAs. They produce,
metabolize, and utilize DAAs [50] [51]. Since microbial DAAs enter the food chain, I
will briefly examine some aspects of the microbiology of DAAs.
Brckner et al. [52] determined the DAA content produced by several classes of
bacteria (Acetobacter, Bifidobacterium, Brevibacterium, Lactobacillus, Micrococcus,
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Fig. 1. Structures of genetically coded amino acids subjected to racemization
Propionibacterium, and Streptococcus) used in starter cultures for the production of
fermented foods and beverages. d-Ala and d-Asp were found at the highest
concentrations in all bacteria. d-Glu was present in several classes of the microorganisms. Lower but significant amounts of d-Leu, d-Lys, d-Met, d-Orn, d-Phe, dPro, d-Ser, d-Thr, and d-Tyr were also detected in some of the bacteria. DAAs in
fermented foods may largely originate from bacteria. Bacteria from oral and intestinal
floras and rumen microorganisms are also potential sources of dietary DAAs [53] [54].
3.2. Peptidoglycans in Bacterial Cell Walls. Bacterial cell walls may constitute a
major source of dietary DAAs. The thick cell wall of a Gram-positive bacterium
consists of peptidoglycan (a sugar – amino acid polymer) and teichoic acid. Both
polymers contain DAAs. The much thinner cell wall of Gram-negative bacteria consists
entirely of peptidoglycan and associated proteins. The composition of the peptidoglycans and teichoic acids may vary among different classes of bacteria. The DAAs in the
bacterial cell walls contribute to their resistance to digestion by proteolytic enzymes.
Related studies showed that:
d-Ala is required for the synthesis of the mucopeptide component of nearly all
bacterial cell walls [55];
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Fig. 2. Structures of post-translational amino acids present in foods and tissues. Amino acids with one
asymmetric C-atom can exists as two, those with two as four, and those with three as eight isomers,
respectively.
the peptidoglycan structure of Lactobacillus casei contained asparagine crosslinks between d-Ala and l-Lys [56];
glutamate racemase in E. coli provides d-glutamate, an indispensable component
of peptidoglycans in bacteria [57];
mutations leading to increased resistance of Enterococcus faecalis to antibiotics
are governed by the amount of d-Ala-d-Ala and d-Ala-d-lactate incorporated into
peptidoglycan precursors [58];
d-Ala-d-Ala contributes to the resistance of antibiotics [59];
the gene encoding d-Thr aldolase (an enzyme that catalyzes the inversion of dThr to d-allo-Thr in E. coli) is responsible for the incorporation of d-Ala into cell wall
teichoic acid in Bacillus subtilis [60] [61];
d-Ser dehydratase from E. coli catalyzes the conversion of d-Ser, d-Thr, and allod-Thr to the corresponding a-keto acids and NH3 [62];
d-Ser inhibits the action of bacterial transaminase, whereas d-Ala partially
protects against this inhibition [63];
d-Asp and d-Glu can serve as indicators for proteins of bacterial origin [64];
anaerobic bacteria are capable of using DAAs and l-sugars for growth [65];
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d-Asp and d-Glu levels correlated with growth of probiotic bacteria during
fermentation in milk and whey powders [66].
3.3. Peptide Antibiotics. DAA-Containing synthetic and natural peptides possess
strong antimicrobial properties. Synthetic peptides that inactivated Clostridium
botulinum include glycyl-d-Ala, myristoyl-d-Asp, sorbyl-d-Ala, sorbyl-d-Trp [67].
DAAs in natural antibiotics include d-Asp and d-Glu (bacitracin, mycobacillin); d-Cys
(malformin); d-Leu (circulin); d-Orn (bacitracin); d-Phe (funigsporin, polymixin,
tyrocidine); d-Ala, d-Leu, d-Val (gramicidin); and d-Ala, d-Leu, d-Val (actinomycin,
valinomycin) [68 – 70]. Substitution of DAAs in the peptides designed to reduce
susceptibility to digestion enhances the activity of pleurocydin [71], bombinins H [72],
lacticin [73], and other antibiotic peptides [74 – 76]. The mechanism of action of these
peptide antibiotics involves the induction of ionic channels that lead to the disruption of
bacterial cell membranes [77].
The cited studies suggest that an understanding of the biochemistry of genes and
enzymes involved in the synthesis and metabolism of DAA-containing functional and
structural microbial molecules that are targets of antibiotics may help efforts designed
to discover improved bactericidal agents.
4. Food Processing-Induced Formation of DAAs. – Historical Perspective. Since the
early part of the 20th century, alkali treatments have been known to racemize amino
acids [78 – 80]. The early authors used changes in optical rotation to determine the
alkali-induced racemization, and Kjeldahl N analysis of NH2 groups of liberated amino
acids and peptides to detect concurrent hydrolysis of peptide bonds. They reported
that:
l-Arg was racemized to the d-isomer, and that the protein clupein was
concurrently partly hydrolyzed and racemized in Ba(OH)2 and NaOH solution at
408 [79];
optically active hydantoins prepared from a-amino acids underwent spontaneous
racemization at room temperature, presumably as a result of keto – enol tautomerism
involving the asymmetric C-atom [78] [81];
racemization of amino acids in gelatin occurred on treatment with 0.1 and 1.0n
NaOH, but not on treatment with 3.0n alkali, presumably because in the latter case the
rate of peptide-bond hydrolysis is higher than that of racemization [80] [82 – 84];
heating casein with 1.0n NaOH at 1258 resulted in complete racemization of all
amino acids, which occurred at a faster rate than analogous treatment of gelatin [83] or
edestin [84].
The field of amino acid racemization in proteins lay largely dormant for
approximately half century because of lack of suitable analytical methods to determine
the formation of specific protein-bound DAAs. The development of GC and HPLC
techniques along with chiral columns that can separate d- and l-isomers stimulated
widespread interest in this subject. The observation by Masters and Friedman [85 – 89]
that widely consumed processed foods contain DAAs stimulated worldwide interest in
the distribution of DAAs in the human diet. They reported the following levels of dAsp in widely consumed foods (d/l ratio; % as d-isomer): coffee-mate (0.208; 17);
Fritos potato chips (0.164; 14); simulated bacon breakfast strips (0.134; 13); Isomil
baby formula (0.108; 10); and textured soy protein meat analogue (0.095; 9). These
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authors were also apparently the first to relate racemization rate constants of proteinbound DAAs to inductive parameters associated with free LAAs (linear free-energy
relationships), and to describe the relationship between DAA content of racemized
casein and in vitro digestibility by proteolytic enzymes [86]. First-time studies by
Friedman and Gumbmann [90] on the biological utilization of DAA as source of the lisomer using all amino acid diets and the predicted formation of cross-linked amino
acids such as histidinoalanine that may accompany racemization [91] that were later
realized are also of historical interest. Some of these aspects are further examined
below.
Racemization Mechanisms. Fig. 3 offers a mathematical derivation of the first-order
kinetic equations that govern reversible in vitro amino acid racemization. Fig. 4 depicts
three different mechanisms that have been postulated to govern racemizations of
amino acids during food processing. Fig. 5 depicts a postulated mechanism for the in
vivo inversion of an LAA to its d-isomer catalyzed by pyridoxal phosphate. Figs. 6 – 9
illustrate the kinetics of racemization, and Fig. 10 (see below) shows susceptibilities of
some amino acids at high pH that may accompany racemization. Tables 1 – 3 list the
DAA contents of racemized proteins. The cited observations indicate that heat and
high pH widely used in processing of food induce the racemization of all amino acid
residues by the indicated pathways.
Alkali-induced peptide-bond cleavage of soy proteins determined by a ninhydrin
assay [35] influenced the extent of racemization [92]. Racemization rates of the same
amino acid in polyamino acids were much lower than in soy proteins. Related studies
[93] showed that:
free amino acids racemized approximately ten times slower than did proteinbound ones;
alkali treatment of zein induced racemization of amino acids residues and caused
nutritional damage without the presence of lysinoalanine (LAL) [94];
serine residues in lactalbumin, leaf protein concentrate, and soy protein were
highly susceptible to heat- and alkali-induced racemization [95];
racemization of protein-bound amino acids, especially of lysine, induced by heat
extrusion was greater in soy than in corn proteins [96];
melanoidins formed during heating of glucose or fructose with LAA or DAA had
similarly shaped but different absorption spectra [97].
5. Distribution of DAAs in Food. – As already mentioned, the observation by
Masters and Friedman [88] that processed commercial foods contained DAAs was
followed by numerous worldwide studies on the content of DAAs in a variety of foods.
The aspect is briefly examined below for several food categories listed in alphabetical
order.
5.1. Alfalfa Seeds. Alanine racemase catalyzes formation of d-Ala in alfalfa seeds
(Medicago sativa L.) [48].
5.2. Animal Skins. Peptides and proteins isolated from animal skin are reported to
contain DAAs [98] [99].
5.3. Bread. Use of lactic acid bacteria and yeast in the fermentation of sourdough
before baking results in the introduction of d-Ala and d-Glu into the dough [100].
Baking of the dough into bread induces a 44% decrease in the total free DAA content.
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Fig. 3. Derivations of first-order kinetic equations for reversible amino acid racemization. Adapted from
[87] [88].
5.4. Coffee. Roasted coffee contained 10 – 40% of d-Asp, d-Glu, and d-Phe [101].
Higher amounts of DAAs were formed during roasting of Robusta than of Arabica
coffees [102]. Liquid coffee contained ca. 20 mg of total DAA/100 ml [103].
5.5. Dairy Products. DAAs in cows milk originate from the digestion (autolysis) of
peptides and proteins containing DAAs originating from microbial cell walls
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Table 1. DAA Content [(d/d þ d) 100) ¼ % d] of Eight Alkali-Treated Proteins. Adapted from
[20] [85] [92] [199] [255].
Amino
acid
Casein
Lactalbumin
Wheat
gluten
Corn
protein
Fish
protein
Soybean
protein
Bovine
albumin
Hemoglobin
Ala
Val
Leu
Ile
Cys
Met
Phe
Lys
Asp
Glu
Ser
Thr
Tyr
LAL a )
15.2
2.6
7.4
3.3
–
24.7
24.4
8.1
29.2
19.7
41.0
29.3
15.0
4.4
14.4
2.7
5.0
3.1
32.1
32.3
24.3
7.2
22.6
19.5
47.1
29.1
18.9
5.4
18.6
4.0
7.2
4.0
32.0
33.1
24.4
9.4
25.6
32.3
42.2
30.0
19.5
0.9
22.2
4.9
7.8
5.5
43.7
29.8
32.4
8.0
41.6
35.0
44.0
36.3
35.5
0.3
19.3
3.1
6.8
3.6
22.8
29.2
28.0
11.5
25.0
18.9
42.1
32.8
16.3
2.8
15.8
2.5
6.3
3.9
21.0
24.3
25.5
11.3
30.8
21.1
44.2
27.8
13.7
3.2
22.1
3.5
8.2
5.7
23.0
30.0
28.1
13.3
27.0
18.4
43.0
28.3
15.3
8.5
17.1
4.0
6.6
5.0
30.0
26.2
30.0
9.9
18.9
19.8
44.5
31.2
22.6
4.4
a
) Mixture of (ld þ ll) LAL isomers in g/16 g N.
Table 2. Variation in LAL Isomer Content of Alkali-Treated Proteins. Adapted from [20] [199].
Protein
LAL [g/16 g N]
[ll-LAL]/([ll-LAL] þ [ld-LAL]), Isomer ratio
Zein
Wheat gluten
Fish protein concentrate
Bovine hemoglobin
Casein
Lactalbumin
Bovine serum albumin
0.32
0.95
2.75
3.36
4.40
5.38
8.52
Not detected
0.50
0.40
0.50
0.51
0.51
0.50
Table 3. Aspartic Acid Racemization and LAL Content of Alkali-Treated Casein and Acetylated Casein.
Adapted from [88].
Protein
d/l Asp
LAL [mol-%]
Casein, untreated
Casein þ alkali
Acetylated casein þ alkali a )
0.023
0.387
0.336
0.00
2.35
0.00
a
) Acetylation of e-NH2 groups of lysine side chains prevents LAL but not d-AA formation.
(peptidoglycan) proteins in the rumen of the cows [104]. Raw milk from ruminants
(cows, goats, and sheep), but not human milk, contains the following DAAs: d-Ala, dAsp, d-Glu, d-Lys, and d-Ser [66] [105] [106]. Relatively high concentrations of these
DAAs were also present in widely consumed ripened cheeses [21]. Emmental cheese
contains as much as 70 mg of total DAAs per 100 g [103]. The DAA content of varied
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Fig. 4. Base-catalyzed racemization: Proton abstraction – addition mechanism. a) An OH ion abstracts
an H þ from the R-CH a-C-atom of an amino acid to form a negatively charged carbanion which has lost
its original asymmetry [256] [257]. The carbanion can then recombine with a proton from the solvent to
regenerate the original amino acid which is now racemic (dl). Elimination – addition mechanism: The
carbanion can also undergo an elimination reaction to form dehydroalanine side chain. The
dehydroprotein can then react with H2O to form racemized amino acid side chains (e.g., dl-Ser)
[85] [258]. The concurrent formation of lysinoalanine is also shown. b) Acid-catalyzed racemization:
Protonation of the carboxy group of an l-amino acid facilitates removal of a proton to form the
dehydroalanine. Proton addition at two sides (re and si faces) of the double bond generates both d- and
l-isomers. Adapted from [259]. c) Racemization of a compound (fructose-l-phenylalanine) intermediate of d-Phe. Adapted from [23].
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Fig. 4 (cont.)
Fig. 5. Isomeric inversion in vivo: catalysis of inversion of an l-amino acid by the pyridoxal phosphate
(PLP) coenzyme of a microbial racemase involving stereoselective shifts of H-atoms. Adapted from [260].
among cheeses and changed during cheese production [107]. Peptide-bond hydrolysis
of casein during storage of cheeses may also influence their DAA content [104].
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Fig. 6. Effect of temperature on content d-Ser, d-Tyr, and LAL of soybean protein. Adapted from
[92] [199] [261].
The cited authors also suggest that:
because the d-Ala content of raw milk increased during storage at 48, d-Ala
could serve as an indicator of contamination by d-Ala-producing psychotropic
bacteria;
because mastitis is an infection of the udder caused by bacteria, milk from
infected cows should have a higher DAA content originating from the microorganisms;
pasteurization of milk does not increase its d-amino content.
5.6. d-Glutamic Acid in Processed Foods. Monosodium glutamate (MSG), the
sodium salt of the naturally occurring nonessential amino acid, is often added at levels
of 0.2 – 0.9% to foods to improve flavor and palatability. A survey showed that a variety
of processed foods contained significant amounts of d-Glu (which does not possess
flavor-enhancing properties) [108]. The d-% ranged from 0.25 for pure MSG to 0.8 for
soups (highest value), 2.9 for crackers, 7.9 for sauces, 18 for vinegars, 36 for sauerkraut
juices, 1.6 for tomato products, and 6.2 for milk products.
The d-Glu could originate from microbial sources as well as from food-processinginduced racemization of free and protein-bound l-Glu. Table 2 shows that proteinbound l-Glu is one of the fastest racemizing amino acids. The possible impact of the disomer of MSG on the Chinese Restaurant Syndrome has apparently not been
evaluated.
5.7. Eggs. Alkali pickling of duck eggs in a 4.2% NaOH/5% NaCl solution for 20 d at
room temperature used to prepare the traditional Chinese pidan resulted in extensive
racemization of amino acid residues and concurrent formation of lysinoalanine (LAL)
[109]. The relative racemization rates appear to be similar to those observed with egg
albumin and other pure proteins.
5.8. Fish. Heating of laboratory-made herring meals at 1258 induced timedependent formation of d-Asp [110]. The d-Asp content of twelve fishmeals from
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various sources ranged from 0.7 to 3.7%. The d-Asp content may indicate the severity
of the thermal treatment during cooking or drying of fish meals [111].
5.9. Food (Dietary) Supplements. Widely used dietary supplements are also
reported to contain DAAs [112] [113].
5.10. Fruits and Vegetables. Fruits (apples, grapes, oranges) and vegetables
(cabbage, carrots, garlic, tomatoes) as well as the corresponding juices contain variable
but measurable amounts of DAAs including d-Ala, d-Arg, d-Asp, and d-Glu
[21] [105]. These amino acids could originate from plant sources, from soil, and other
microorganisms, and/or from heat treatments used to kill pathogens.
5.11. Fruit and Vegetable Juices. Brckner and Westhauser [114] reported the
following total DAA amounts in fruit and vegetable juices (in mm): carrot juice, 8.5;
orange and other fruit juices, 28 – 57; tomato juice, 14.5. Other investigators [22] [27]
confirmed that DAAs occur naturally in fruit juices, beers, and wines. Specific DAAs
could permit differentiating juices from biologically dissimilar fruits, and could serve as
an indicator for detecting bacterial activity and shelf-life of fruit juices.
5.12. Honey. The d/l ratios of Leu, Phe, and Pro could serve as indicators of age,
processing, and storage histories of honeys [115].
5.13. Maize (corn grain). Significant differences were observed in the DAA content
of some transgenic maize (corn) cultivars compared to standard varieties [36]. The
authors suggest that this finding opens up new approaches in comparing the
composition of transgenic plant foods to their conventional counterparts.
5.14. Meat Products. Low levels of DAAs (1.25 – 13.79 mg/g) were detected in both
irradiated and non-irradiated cooked hams [116]. There was no correlation between
radiation dose and DAA content.
5.15. Spices. Liquid spices contain very high amounts of DAAs, up to 600 mg/100 ml
[103].
5.16. Vinegars. Several investigators reported that fermentation conditions used to
prepare different vinegars (balsamic, cider, sherry, white wine) affect their DAA
profiles [117 – 120].
5.17. Wines. Autolysis of yeast (Saccharomyces cervisiae) produces compounds that
beneficially influence the quality of sparkling wines. Giuffrida et al. [121] found that the
pattern of release of DAAs from conventional yeast strains differ from transgenic ones.
The content of several DAAs of 26 wines did not correlate with storage times [122].
Wine marinades can be used to inactivate bacteria [123] that may produce DAAs.
Other potential dietary sources of naturally occurring DAAs include fungi, insects, fish,
and other marine organisms [13].
6. Nutrition, Pharmacology, and Toxicology of DAA. – Twenty naturally occurring
amino acids and several hundred amino acid derivatives formed in vivo posttranslationally in plants and animals, and in vitro during food processing play a
fundamental role in nutrition and other aspects of animal and human health. The
general requirements are adequate amounts of the essential amino acids with a
reasonable balance among all [124]. Bioavailability and biological utilization of both
LAAs and DAAs vary widely and depend on source, chemical and metabolic
interactions, and on the diet and health of the consumer. To develop a better
understanding of the nutritional significance of DAAs and alkali-treated food proteins,
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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Fig. 7. Relationship between racemization rates (k) relative to Ala, and the inductive constant (s*) of the
amino acid side chain R in RCH(NH2 )COOH. Adapted from [199] and [93].
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
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Fig. 7 (cont.)
we compared the weight gain in mice fed free amino acid diets in which the test LAA
was replaced with the d-isomer. The results obtained reflect the ability of mice to
utilize DAA in the complete absence of the l-form. In the case of essential amino acids,
the mice must meet the entire metabolic demand from the d-isomeric forms.
Biological utilization by weanling male mice of DAAs was tested in a 14-day growth
assay by using an all-free amino acid diet in which each l-amino acid was replaced by
the d-isomer. The growth assay was standardized with six mice per group, with potency
estimation being based of response of two to seven groups. Statistical evaluation of
potencies of the DAA was calculated as the slopes of growth curves (weight gain after
14 d per unit of dietary concentration) where linearity was approximated. Mean body
weight gains were compared by Duncans multiple range test using individual values.
The results illustrated in Figs. 10 and 11, and Table 4 show wide variations in the
utilization of DAAs as a nutritional source of the corresponding l-isomers.
Digestion and Nutrition. A number of studies described the adverse and beneficial
effects of alkali treatment on protein nutrition [86] [125 – 132]. Friedman et al. [86]
attempted to quantitatively define the susceptibility of alkali-treated casein to in vitro
digestion by trypsin and chymotrypsin (Fig. 11, a). Monitoring on a pH-stat revealed an
approximately inverse relationship between DAA and LAL content on one hand and
the extent of proteolysis measured on the other. The decreased susceptibility of alkalitreated casein to digestion could arise from loss of susceptible sites to enzyme cleavage.
Sarwar and Paquet [133] reported that, although tripeptides composed of LAAs were
completely hydrolyzed by intestinal mucosal peptidases, the following DAA-containing
tripeptides largely resisted digestion: Ala-d-Met-Ala, Val-d-Met-Phe, Ala-d-Glu-Ala,
Val-d-Asp-Ala, and Ala-d-Phe-Leu. Rat feeding studies also showed that d-Met in the
tripeptides was less available for growth than l-Met. Alkali treatment by Jenkins et al.
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Fig. 8. Arrhenius Plots for the racemization of protein amino acid residues in proteins: a) casein; b) soy
protein. Adapted from [85] [199].
induced formation of DAA and nutritional damage in corn proteins (zein) without
formation of LAL [94].
To obtain additional information on this aspect, we evaluated nutritional and
histopathological consequences of feeding-toasted and alkali-treated soy flours to
baboons. Fig. 11, b, shows the growth curves for seven pre-adolescent male baboons
each fed soy control and alkali-treated soy diets. There was no difference in growth
rates over a 150-day period, since the slopes of the growth curves are not significantly
different from each other. However, in absolute terms, weight gain of the baboons fed
the treated soy diet (containing 370 mg of LAL/100 g air-dry diet) was ca. 20% lower
than the gain observed with the control soy diet (containing 80 mg of LAL/100 of air-
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Fig. 9. Effect of temperature on DAA content in coffee. Adapted from [102].
dry diet). The decreased weight gains may be due to the decreased digestibility and
utilization of the alkali-treated soy protein compared to the control.
Using a 15N-tracer technique, Heine et al. [134] investigated the utilization of DAAs
by infants fed parenterally. Retention of 15N in the protein pool ranged from 23.2% for
d-[15N]Val to 48.6% for d-[15N]Ala. DAAs were utilized by the infants for protein
synthesis only to some and variable extent.
Possible causes for the reduction in digestibility – nutritional quality include
destruction of proteolytic enzyme substrates such as Arg and Lys, isomerization of
LAAs to less digestible d-forms, formation of inter- and intramolecular cross-links that
hinder access of proteolytic enzymes, inhibition of proteolytic enzymes [135 – 139], and
alkali-induced formation of trypsin- and chymotrypsin-inhibiting peptides [132].
7. Pharmacology, Toxicology, and Potential Medical Uses of Individual DAA. – To
facilitate understanding the complex nature of biological activities of DAAs, we briefly
summarize results of published studies on the following nutrition- and health-related
aspects of DAA.
7.1. d-Ala. Because it is part of the structure of bacterial cell walls, d-Ala derived
from microbial sources is present in many foods as well as in hydrolyzed protein
fertilizers [25]. d-Ala is structural component of bacterial cell membranes [140] [141]
and is a possible indicator of bacterial contamination, heat treatment, and shelf life of
fruit juices and other foods [22] [105]. It can also be used in studies of molecular
imaging [142], to induce cytotoxic oxidative stress in brain tumor cells [143], and to
treat schizophrenia [144]. Changes in d-Ala content of the rat pancreas are related to
their diurnal and nocturnal (circadian) habits [145]. The submandibular gland and oral
epithelial cells, not ingested food or oral bacteria, appear to be the source of high levels
of d-Ala and d-Asp in human saliva [146].
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Fig. 10. Effect of pH on the extent of degradation of susceptible amino acids. Adapted from [89].
7.2. d-Arg. Both l- and d-Arg protected against oxygen-radical-induced injury of
rat heart tissue [147] and against endotoxin shock in rabbits [148]. d-Arg and other
DAAs inhibited cell proliferation and tumor growth in rats [149], acted as a central
nervous system stimulant, and exhibited anticonvulsant activity in humans [150].
7.3. d-Asp. d-Asp aggravated the nephritis in rats induced by Staphylococcus aureus
bacteria [151] and prevented K and Mg depletion in rats induced by diuretics [152].
7.4. d-Cysteine (d-CysSH). Although l-CysSH had a sparing effect of l-Met
consumed by mice, d-CysSH did not [153]. In fact, d-CysSH imposed a metabolic
burden as indicated by depressed growth when fed to mice with less than optimal levels
of d-Met (Fig. 12, a). The 24% decrease in weight gain of the d-CysSH plus l-Met
amino-acid diet compared to l-Met alone implies that d-Cys is nutritionally
antagonistic or toxic.
d-CysSH but not N-acetyl-d-cysteine lowered rat blood cyanide levels derived from
acrylonitrile [154]. d-CysSH is also reported to be involved in the detoxification and/or
prevention of toxicities caused by cyanides [155], the drug paracetamol [156], and
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
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Fig. 11. Nutritional utilization of alkali-treated proteins by mice and baboons. Adapted from [262].
other drugs [157 – 159]. l-Cysteine-glutathione disulfide, but not the d-cysteine analog,
protected mice against acetaminophen-induced liver damage [160]. d-Cysteine
desulfhydrase catalyzes the elimination of H2S form d-cysteine, presumably to form
dehydroalanine [161].
7.5. d-Cystine (d-Cys). Although l-Cys is not an essential amino acid for rodents,
less l-Met is needed for growth if the diet contains l-Cys [153]. Our results also show
that l-Cys is somewhat more efficient in sparing d-Met than in sparing l-Met in diets
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Table 4. Growth Responses to DAA and Peptides in Mice Fed Amino-Acid Diets. Adapted from
[90] [153] [172] [219 – 221] [255].
DAA and peptides
Relative nutritional value to l-form [%]
d-Amino acids
d-Methionine
d-Phenylalanine
d-Tryptophan
d-Leucine
d-Histidine
d-Valine
d-Threonine
d-Isoleucine
d-Lysine
l,l þ d,l-Lysinoalanine
79.5
51.6
24.7
12.4
8.5
5.1
3.1
1.2
8.2
3.8
Peptides and sulfoxides
N-Acetyl-l-methionine
N-Acetyl-d-methionine
l-Methionyl-l-methionine
l-Methionyl-d-methionine
d-Methionyl-l-methionine
d-Methionyl-d-methionine
l-Methionine sulfoxide
d-Methionine sulfoxide
N-Acetyl-l-methionine sulfoxide
N-Acetyl-d-methionine sulfoxide
l-Methionine hydroxyl analogue
d-Methionine hydroxyl analogue
89.6
22.9
99.2
102.9
80.4
41.0
87.5
26.8
58.7
1.7
55.4
85.7
containing low levels (0.29%) of the two isomers (Fig. 12, b). Supplementation of dMet with an equal sulfur equivalent of l-Cys doubled growth. Thus, the overall
response was equal to that produced by l-Met in the presence of l-Cys. In contrast,
supplementation of suboptimal levels of l-Met with increasing concentrations of d-Cys
reduced the growth rate of mice. Excess d-Cys in the diet is toxic.
7.6. Dehydroalanine. Dehydroalanine is an intermediate in the racemization of
protein-bound amino acids (Fig. 4). We found that its content (in g/16 g N) in alkalitreated casein was 0.33 and in alkali treated acetylated casein 1.39 [162]. Dehydroalanine can, in principle, act as a biological alkylating agent similar to that suggested for
processing-induced acrylamide [163] [164].
7.7. d-His. d-His enhanced Zn accumulation and reduced the fraction of Zn that was
retained and absorbed by fish [165]. Both d- and l-His enhance DNA degradation by
H2O2 and Fe2 þ ions [166]. d-His-induced cell injury is mediated by an Fe-dependent
formation of reactive oxygen species [167] [168].
7.8. d-Homocysteine. Both d- and l-homocysteine inhibit transmethylations and
prevent neural tube defects in rat embryos [169] [170].
7.9. Lanthionine (LAN) Isomers. LAN Isomers are formed during the biosynthesis
of microbially derived peptide antibiotics [171] and during exposure of proteins to
alkali and heat. Reaction of the SH group of CysSH and the double bond of
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Fig. 12. Effects of d-amino acids added to mice diets. a) Sparing effects of l- and d-Cys, and other sulfur
amino acids in methionine-deficient diets. b) Effect of d-cystine in low methionine diets. c) Sparing
effect of d-Tyr on growth of mice on l-Phe-deficient diets. d) Effect of d-Tyr on growth of mice on highand low-protein diets. Adapted from [90] [153].
dehydroalanine gives rise to one pair of optically active d- and l-isomers, and one
diastereoisomeric (meso) form of LAN [33]. The mixture of dl þ meso-LAN has a
sparing effect on l-Met, as evidenced by a 27% greater weight gain when the two
amino acids were fed together, than when fed suboptimal l-Met [153] [172]. Base-
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catalyzed formation of LAN and LAL does not affect bioactivity in bovine
somatotropin [173].
7.10. d-Lys. d-Lys is not utilized as a nutritional source of l-Lys by chicks, dogs,
mice, rats, or humans, presumably because d-amino acid oxidase does not metabolize
d-Lys [174]. The extent of nutritional damage of alkali treatment associated with loss of
lysine may depend on the original lysine content of a protein. Decrease in lysine due to
racemization and LAL formation in a high-lysine protein such as casein, high-lysine
corn protein, or soy protein isolate may have a less adverse effect than in a low-lysine
protein such as wheat gluten or corn protein, where lysine is a nutritionally limiting
amino acid [175] [176]. Because of its low toxicity, d-Lys may be a better candidate to
reduce radioactivity uptake by the kidneys during cancer therapy with radionuclides
than is l-Lys [177 – 179].
7.11. Lysinoalanine (LAL) Isomers. The cross-linked amino acid LAL, formed
concurrently with DAA in alkali-treated proteins, has two asymmetric C-atoms, making
possible four diastereoisomeric forms: dd, dl, ld, and ll. The nutritional value of the
individual isomers as a source of l-Lys is not known. Table 2 shows that a mixture of lland ld-isomers has a value for the mouse equivalent on a molar basis to 3.8% of l-Lys.
We have examined the affinity of LAL towards a series of metal ions, of which CuII
was chelated the most strongly. The four LAL isomers differ in their ability to chelate
metal ions such as Cu [180] [181]. On this basis, we suggested a possible mechanism for
kidney damage in the rat involving LALs interaction with Cu within the epithelial cells
of the proximal tubules. The direct relationship between the observed affinities of the
two LAL isomers for CuII ions in vitro and their relative toxic manifestation in the rat
kidney suggests that LAL exerts its biological effect through chelation of Cu in body
fluids and tissues. The observed binding of ll- and ld-LAL to CoII, ZnII, and other
metal ions imply that LAL could also influence Co utilization.
LAL, ornithinoalanine (OAL), and LAN are formed during alkaline treatment of
wool during skin unhairing of sheep and other processes designed to enhance the
strength of wool, and possibly also human hair, via cross-linking reactions [91] [182].
Serine racemase contains a catalytically active LAL residue [183].
7.12. d-Met. The nutritional value of d-Met in mice approaches that of l-Met
(Table 2). By contrast, d-Met appears to be poorly utilized by humans when consumed
either orally or during total parenteral nutrition. One factor giving rise to inconsistencies in the utilization of d-Met is the dose dependency of the apparent potency of
d-Met relative to its l-isomer, i.e., the dietary level of the d-form for any given growth
response relative to that of the l-form that would produce the same growth response.
This dose-dependency is a result of the non-linear nature of the dose – response curves
(Fig. 13). This complicates attempts to compare results from mice with those of other
animal species. The latter studies often provided data based on a single substitution of
the d- for the l-isomer. The figures show that high levels of l-Met (but not of d-Met)
are toxic since they inhibited growth of mice [184].
d-Met-Containing solutions inhibited tumor cell growth in vitro [185], and
protected against cisplatin ototoxicity in humans [186 – 189] and against oral radiation
[190]. A cascade of enzymes transformed d-Met to the l-form [191].
7.13. Orn Isomers. Martnez-Girón et al. [113] [192] developed analytical methods
for the separation of unnatural ornithine isomers that may be present in processed foods.
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7.14. d-Phe. The relative growth rate of mice fed d-Phe replacing the l-isomer in a
free-amino-acid diet is concentration-dependent, ranging from 28.3 to 81.3% when
compared to control diets containing the same amounts of l-Phe (Figs. 12 and 13, and
Table 4) [90]. The data suggest the absence of any antinutritional effects or toxicity
from feeding either Phe isomer at twice the optimum dietary level. d-Phe and
hydroxyproline were reported to define the native structure and presumably virulence
of conotoxin, a toxic peptide obtained from the venom produced by cone snails [193].
7.15. d-Pro. Oral feeding of an aqueous solution of d-Pro for one month to rats
induced fibrosis and necrosis of kidney liver cells and elevation of serum enzymes
[194]. By contrast, orally fed d-Pro and d-Asp induced did not induce acute toxicity in
rats [195].
7.16. Selenomethionine Isomers. Selenomethionine is a major source of Se in the
diets of both animals and humans. Comparison of the effects of oral consumption of
seleno-l-Met, seleno-dl-Met, and selenized yeast on the reproduction of mallard
ducklings revealed that, although both seleno-Met preparations were of similar toxicity,
their potency was greater than that of Se present in yeast [196]. Heinz et al. [196] found
that the survival of day-old ducklings consuming l-seleno-Met after two weeks was
significantly lower (36%) than that of ducklings consuming the dl-isomer (100%). dseleno-Met protected against adverse effects induced by space radiation [197] [198].
7.17. d-Ser. Protein-bound l-Ser racemizes faster to the d-isomer than the other
amino acids (Table 1, and Figs. 6 and 7) [199]. d-Ser has been reported to enlarge rat
kidney cells (cytomegaly) similar to that observed with LAL. Several approaches were
used in an attempt to elucidate possible mechanisms of d-Ser renal toxicity [200 – 202].
Sodium benzoate [200] [201], protein-deficient diets [203], and a-aminoisobutyric acid
[204] attenuated d-Ser nephrotoxicity in rats.
d-Ser-induced kidney damage may be due to a lowering of the concentration of
renal glutathione (GSH) that protects the kidneys against kidney-damaging reactive
oxygen species [204]. The decrease in glutathione concentration takes place during the
metabolism of d-Ser by DAA oxidase. Because nephrotoxicity induced by d-Ser is
similar to that caused by LAL, the question arises as to whether effects of these two
amino acids on the rat kidney are competitive, additive, or synergistic?
Because d-Ser also acts as an agonist of N-methyl-d-aspartate (NMDA) receptormediated neurotransmission in the brain, it may be useful as innovative pharmacologic
strategy in schizophrenia and pain [205]. A strain of protozoa (Drosophila
melanogaster) has developed a transport mechanisms with adaptive specialization in
the absorption and brain functions of d-Ser and other DAA that may be involved in
neuronal functions [206]. d-Ser can be enzymatically transformed to l-Trp [207]. The
d-configuration of Ser maintains the toxic conformation of the mushroom poison
viroisin [208].
7.18. d-Thr. l-Thr is the second-limiting amino acid in maize (corn) proteins. The
utilization of d-Thr by the chick, rat, mouse, or human as a nutritional source of the lisomer is insignificant [174].
7.19. d-Trp. The relative nutritional potency of d-Trp compared to l-Trp in mice is
strongly dose-dependent, being inversely related to the dietary concentration and
ranging from 29 to 64% (Fig. 11) [209]. The maximum growth obtainable for l-Trp
occurred at 0.174% in the diet. By increasing the dietary concentration of d-Trp up to
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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Fig. 13. Top four plots: weight gain in mice fed increasing dietary levels of l-Met, d-Met, and isomeric
methionine derivatives and analogs for 14 d. Asterisk indicates significant difference from l-Met at the
same dietary concentration (top four plots). Adapted from [153] [172]. Lower two plots: relationships of
weight gains to percent of l- and d-Phe, and l- and d-Trp isomers in amino acid diets fed to mice. Adapted
from [90] [209] [216].
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
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Fig. 13 (cont.)
0.52%, growth passed through a maximum at 82% of that achieved with the l-isomer.
This occurred at 0.44% of d-Trp.
Considerable species variation exists for the nutritive value of d-Trp [174]. In
chicks diets, relative potency of the d- to the l-isomer has been reported to be 20%. dTrp was well-utilized by growing pigs [210]. The value for humans is ca. 10%. Rats
utilize d-Trp as efficiently as l-Trp. d-Trp can serve as a niacin precursor in rats to the
same extent as does the l-form [211] [212]. These studies also showed that d-Trp has
one sixth of the activity of niacin. Both d-Phe and d-Trp taste sweet [209] [213 – 215].
7.20. d-Tyr. Nutritionally, l-Tyr is classified as a semi-essential amino acid [7].
Combinations of l-Tyr and l-Phe are complementary in supporting growth of mice
[90]. Thus, under conditions where l-Phe may be limiting, l-Tyr may supply half the
requirement of l-Phe alone for chicks, mice, rats, and humans. Our mice-feeding
studies showed that with d-Tyr in an amino acid diet, growth inhibition was severe at a
d-Tyr/l-Tyr ratio of 2 : 1, but was less so when the ratio was 1 : 1 (Fig. 12, c). Similar
results were obtained with a casein diet supplemented with d-Tyr (Fig. 12, d). The
antimetabolic manifestation of d-Tyr may be ascribed to interference with the
biosynthesis of vital neurotransmitters and proteins in vivo [90] [216].
d-Tyr has no sparing effect for l-Phe in mice. Growth inhibition may become
evident when d-Tyr is present in the diet at a level equal or greater than l-Phe. The
potential for chronic toxicity following exposure to lower levels of d-Tyr remains
unknown. Formation of d-tyrosyl-tRNATyr may be responsible for the toxicity of d-Tyr
toward E. coli [217].
7.21. d-Val. Administration of total parenteral nutrition (TPN) containing d-Leu,
d-Met, d-Phe, and d-Val to hepatoma-bearing rats showed that d-Val inhibited tumor
growth without negative effects on the host. d-Leu and d-Met also improved the
nutritional status of the sick rats [218]. These observations suggest that some DAA
diets may benefit cancer patients.
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In summary, mice provide a good animal model to study the biological utilization
and biological effects of DAAs, both free and protein-bound. A major advantage of
mouse bioassays is that they require approximately one-fifth of the test substance
needed for rats and can be completed in 14 days [90] [153] [172] [219 – 221].
8. Role of DAA in Humans. – Indicators of Aging. There appears to be a direct
relationship between the age of the collagen proteins in rat teeth and their d/l-Asp or
d/l-hydroxyproline ratios, presumably because of little or no metabolic activity in the
molar teeth after their formation [222] [223]. The extent of Asp racemization can also
be used to estimate the age at death [224 – 226]. The use of spontaneous racemization of
l- to d-Asp and of other amino acids to assess the age of tissues such as dentin, bone,
and eye lens [227 – 231] has been questioned by Clarke and co-workers [232] [233].
Their objection is based on their findings that red-cell protein aspartyl/asparagine
residues racemize much more slowly in erythrocyte and other proteins than in proteins
of the lens, tooth enamel, and dentin. The in vivo rates of d-Asp formation in
erythrocyte proteins were similar to the in vitro rates. It is not known whether
biochemical processes exist to degrade or repair deamidated and isomerized aspartyl
and asparagine residues in tissue proteins. However, Chinese scientists [14] reported
that human d-Tyr-tRNA (Tyr) deacylase protected cells and neurons against adverse
effects of excess DAA by deacylating d-aminoacyl-tRNAs into free DAA and tRNA.
The in vivo formation of d-Asp may not be the result of a racemization process for
which the d/l ratio cannot exceed 1.0, but rather of an inversion of configuration of lto d-Asp, where the ratio can and does exceed 1.0 [234]. Age-related accumulation
of both DAAs and advanced glycation end products, and the decreased functional capacities of structural and bioactive proteins contribute to the development
of diabetic complications and eye diseases [235]. Moreover, because the accumulation of d-Asp during aging has been implicated in the pathogenesis of Alzheimers
disease, arteriosclerosis, and cataracts, the question arises as to whether the enzyme daspartyl endopetidase reported to degrade d-Asp-containing peptides and proteins in
mammalian mitochondria and nuclei [236] can slow down or reverse the aging of
tissues.
Role of DAA in Disease. Changes in the concentrations of DAAs may be related to
the pathogenesis and therapy of diseases [237]. The total content of d-Phe and d-Tyr
was significantly greater in patients suffering from chronic renal failure than in normal
humans [238]. The increase in DAAs in renal failure may arise from preferential
retention of DAAs in the kidneys (low glomerular filtration compared to l-isomers),
depletion, and inhibition of DAA oxidase activity, and the consumption of DAAcontaining foods and antibiotics. High levels of d-Ser and d-Pro are also reported to
accumulate during renal failure [239]. Down-regulation of energy metabolism after dSer treatment may be related to the mechanism of its nephrotoxicity [240].
The free d-Ser in normal human brain tissue (d/l ratio 0.086) was comparable to
that in Alzheimers-affected human brain (d/l ratio 0.099). Because d-Ser (like the lisomer) passes through the blood – brain barrier, d-Ser in the brain probably originates
from the diet. d-Ser is metabolized by d-amino oxidase in the brain. In contrast, other
studies suggested that synthesis of d-Ser in the brain occurs by in vivo racemization of
l-Ser [241]. These conflicting suggestions need to be resolved in view of the fact that,
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
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although at high pH protein-bound l-Ser racemizes rapidly (Table 2), the amount of dSer in food proteins that have not been exposed to alkaline conditions are too low to
account for the relatively high amounts detectable in the brain.
An understanding of d-Ser signaling in the human brain may facilitate development
of therapies for brain disorders [242]. In contrast to the apparent beneficial role in
neurotransmission [16] [17], d-Ser concentrations above physiological levels induced
lipid and protein oxidative damage and decreased glutathione levels in brain cortex of
rats [243]. d-Ser availability in the nervous system may be altered in schizophrenia
because of increased DAA degradation by DAA oxidase [244].
The involvement of d-Ser in neurotransmission is supported by a double-blind,
placebo-controlled trial with 31 schizophrenic patients. This study revealed that d-Ser
(30 mg/kg/day) added to an antipsychotic regimen had significant beneficial effects on
cognitive function and performance [245]. The d-Ser was well tolerated by the patients
with no apparent side-effects. Will protein-bound d-Ser induce similar improvements?
The following additional observations suggest that DAAs may benefit disease
therapy:
d-Ser can contribute to the therapy of post-traumatic stress disorder [246];
DAA oxidase oxidizes d-DOPA in the brain, which may then be converted to
dopamine into an alternate biosynthetic pathway for the treatment of Parkinsons
disease [247];
DAA inhibit tumor growth in rats, and may be useful in cancer gene therapy
[143] [248];
Parenteral administration of d-Val hepatoma-bearing rats induced significant
improvement in nutritional status and tumor-inhibition compared to control diets
[249];
d-Phe induces analgesia in mice and humans [250];
The observed clinical relief in chronic-pain patients given oral doses of 750 to
1000 mg of d-Phe is ascribed to inhibition of carboxypeptidase and the accompanying
increase in the levels of brain enkephalin. Whether dietary levels of d-Phe present in
processed food proteins can induce analogous pain relief merits study.
Although healthy young adults can invert approximately one third of d-Phe to its lisomer [251], and human plasma contained high concentrations of d-Ser, d-Ala, and dPro (d/l ¼ 0.24, 0.21, and 0.1, resp.), no DAAs were present in plasma proteins
[155] [252]. The total DAA content of the plasma correlated with the serum creatinine
levels. The possible significance of high levels of DAAs in human saliva for oral health
is not known [146]. The possible value of d-Tyr in treating hypertension [253] and the
possible significance for human reproduction of the observed high content of d-Asp in
rat testis fluids and tissues, and spermatozoa [254] are also not known. It appears that
DAAs possess both adverse and health-promoting attributes. The challenge is to
optimize the beneficial ones.
9. Conclusions and Outlook. – There is a need to standardize methods designed to
ascertain the role of DAAs in nutrition. Because the organisms are forced to use the
DAAs as the sole source of the l-forms, the use of all-amino-acid diets in which the lisomer is completely replaced with different levels of the corresponding DAA may be
preferable to supplementation of proteins with DAA. DAAs along a peptide chain may
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be less utilized than the l-forms. The utilization of any DAA may be affected by the
presence of other DAA in the diet. Largely unresolved are the following questions:
Can analytical methods be developed for all four possible isomeric forms (ll, ld,
dd, and dl) of cross-linked amino acids with two asymmetric C-atoms (Fig. 2).
How do biological effects of DAAs vary, depending on whether they are
consumed in the free state or as part of a food protein?
Can poorly digestible racemized food proteins serve as dietary fiber in the
digestive tracts of humans?
Do DAAs and d-peptides alter the normal microflora of the intestine?
Investigate differences in the interactions of DAAs as compared to LAAs in the
binding to the active sites of proteases, such as trypsin, chymotrypsin, and pepsin in the
digestive tract.
Do metabolic interactions, antagonisms, or synergisms among free and proteinbound DAAs occur in vivo?
Do proteins and peptides acquire antibiotic competence upon racemization?
Does racemization alter protein conformations and charge distributions (isoelectric points) resulting in beneficial protective effects against protein-induced allergy,
celiac disease, and bacterial, plant, and venom toxic proteins?
Does food processing-induced formation of DAAs adversely or beneficially
affect the safety of concurrently formed potentially toxic acrylamide?
Does pasteurization and other heat treatments widely used to kill pathogenic and
spoilage bacteria and fungi in liquid (milk, fruit juices) and solid (meat) foods result in
reduced levels of DAAs produced by the foodborne microorganisms?
Will DAAs prevent biofilm formation by bacteria on or in contaminated food
[263]?
I thank Patricia M. Masters (Scripps Institution of Oceanography, La Jolla, CA), Remy Liardon
(Nestle Research Center, CH-Lausanne), Kevin Pearce (New Zealand Dairy Research Center, Palmerston
North), and Michael R. Gumbmann (this laboratory) for excellent scientific collaboration, and Carol E.
Levin for assistance with the preparation of the manuscript. I am grateful to Professor Hans Brckner for
his invitation to submit a contribution to this Topical Issue on d-amino acids.
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